Bacterial cells respond to the changes in their chemical environment (chemotaxis) by binding of the chemical ligand to membrane receptors, activating the multi-protein receptor signaling complex, and generating a diffusible intracellular signal that ultimately regulates the rotation of the flagella motor. The chemotactic signaling pathway in E. coli has emerged as the best-characterized signal transduction network in biology. All the protein components responsible for excitation and adaptation have been identified and characterized, and their soluble domain structures determined to atomic resolution. There have also been numerous mutagenesis, chemical cross-linking and GFP-tagging studies on chemotaxis signaling. However, the structures of the basic signaling unit consisting of the receptor, the kinase CheA and coupling protein CheW, that are essential to understand the molecular mechanism of the signal transduction remain elusive. The studies described in this proposal are targeted to obtaining high resolution structures of the ternary receptor signaling complex, as well as structures of its higher order assembly in intact cells, primarily by high resolution three-dimensional cryo- electron microscopy. We will also characterize the conformational changes of the complex upon receptor activation/inactivation using both structural methods and functional assays. These structures, combining with functional and biochemical analysis, are expected to provide a detailed understanding on how minute external chemical signals are transmitted to the histidine kinase and amplified through a large assembly of signaling complexes within the native cells. These results will also provide a foundation for generating computational models of receptor signaling systems, since the E. coli chemotaxis has been an ideal model system for understanding the molecular mechanisms of signal transduction and signal processing in general. PUBLIC HEALTH RELEVANCE: The mechanism of stimulus-response coupling in bacterial chemotaxis has emerged as a paradigm for understanding the principles of intracellular signal transduction both in bacterial and eukaryotic cells. E. coli chemotaxis is also a representative of the highly conserved "two-component systems" that control processes ranging from cell differentiation and development to circadian rhythms and pathogenesis in prokaryotes including both eubacterial and archaeal species. These systems all contain two central enzymes, a histidine protein kinase and a response regulator that mediates phosphor relay signal transduction networks in microorganisms and plants. In addition, bacterial chemotaxis response is crucial for colonization and infection, and the signal transduction systems that mediate such responses are potential targets for antimicrobial drug development. A deeper understanding of the mechanism of signaling in bacterial chemotaxis is therefore of great interests for many areas of biology and medicine. Our proposed efforts for a comprehensive and integrated structural and functional analysis of chemotaxis receptor signaling complexes and complex arrays will provide insight into receptor-kinase interaction, signaling complex formation, sensory cluster formation and conformation coupling, and contribute to a better understanding of the signal transduction and signal processing in general.